Rapid neurotransmitter uncaging in spatially
Shy Shoham1,2,4,5, Daniel H O’Connor1,2,5, Dmitry V Sarkisov2,3& Samuel S-H Wang1,2
Light-sensitive ‘caged’ molecules provide a means of rapidly and
noninvasively manipulating biochemical signals with submicron
spatial resolution. Here we describe a new optical system for
rapid uncaging in arbitrary patterns to emulate complex neural
activity. This system uses TeO2acousto-optical deflectors to
steer an ultraviolet beam rapidly and can uncage at over 20,000
locations per second. The uncaging beam is projected into the
focal plane of a two-photon microscope, allowing us to combine
patterned uncaging with imaging and electrophysiology. By
photolyzing caged neurotransmitter in brain slices we can
generate precise, complex activity patterns for dendritic
integration. The method can also be used to activate many
presynaptic neurons at once. Patterned uncaging opens
new vistas in the study of signal integration and plasticity
in neuronal circuits and other biological systems.
Neurons integrate synaptic signals from many thousands of
inputs. Understanding the resulting information processing is a
central theme in experimental and computational neuroscience.
Multiple inputs can interact to amplify1, attenuate2or modulate
one another. Thus, results obtained by activating one or a few
inputs at a time, as is commonly done in brain slice experiments,
do not fully capture the complexity of signal processing by
This potential complexity of signal integration suggests the need
to manipulate biochemical and electrical events across dendrites
and in multiple neurons with fine spatial and temporal resolution.
An attractive option for performing such manipulations is the use
neurotransmitters and second messengers3,4. In optical uncaging, a
biologically active molecule is inactivated through covalent attach-
ment of a caging group, is introduced into tissue and is then
converted to its active form by a flash of light. A large variety of
caged compounds is available3, including agonists for neurotrans-
mitter receptors such as glutamate, GABA, acetylcholine and
biogenic amines and intracellular messengers such as calcium
(in which a chelator of calcium is the molecule caged) and
inositol-1,4,5-trisphosphate (IP3). Uncaging approaches open the
possibility of using light to probe semi-intact tissue noninvasively.
Caged compounds are useful whenever control of cellular
biochemistry is needed on subsecond time scales. In addition to
applications to neurophysiology, caged compounds have been
useful in studying other biological problems requiring comparable
time resolution such as secretion5, muscle activation6, fertilization7
and nuclear signaling8. An intriguing recent advance is the devel-
opment of a caged inhibitor of protein synthesis9.
Uncaging light pulses are usually delivered to a single fixed
location. To overcome this limitation we have developed a system
that rapidly deflects and modulates the uncaging beam using
acousto-optical deflectors (AODs). AODs allow extremely rapid
access to many locations in tissue and have been used previously
in some commercial (Noran Inc.) or custom-built10,11imaging
systems. Our system uncages at over 20,000 locations per
second, considerably faster than older uncaging systems that
steer beams with modified galvanometer mirrors and mechanical
shutters12,13. Here we present the basic physical characteristics
of this system and demonstrate its application to several problems
General system design
Oursystem designisillustratedin Figure 1aanddescribedindetail
in the Supplementary Note online. The ultraviolet light source is
a frequency tripled Nd:YVO4 laser (DPSS Corp.; 50–60 ns,
l ¼ 355 nm pulses at a 100-kHz repetition rate with average
power 4400 mW) whose beam is expanded threefold in diameter
and directed through two crosswise-oriented tellurium dioxide
(TeO2) AODs and a two-lens 1:1 telescope into the optical path
of a two-photon microscope14. The AOD assembly, lenses and an
iris are spaced at intervals approximately equal to the lens focal
beam that pivots around the AOD axis to a pivot point at the back
aperture of the objective, thus allowingscanningof the beam in the
focal plane15. The laser output is gated using its Q-switching
control so that output pulses are emitted only after the AOD has
settled at a new value. Control pulses set the repetition rate of the
laser to be the same as the AOD switching rate. At the resulting
pulse rates the energy per pulse is reproducible (s.d. divided by
RECEIVED 25 MAY; ACCEPTED 22 AUGUST; PUBLISHED ONLINE 21 OCTOBER 2005; DOI:10.1038/NMETH793
1Department of Molecular Biology,2Program in Neuroscience and3Department of Physics, Lewis Thomas Laboratory, Washington Road, Princeton University,
Princeton, New Jersey 08544, USA.4Present address: Faculty of Biomedical Engineering, Technion 32000, Haifa, Israel.5These authors contributed equally to this work.
Correspondence should be addressed to S.S.-H.W. (firstname.lastname@example.org).
NATURE METHODS | VOL.2 NO.11 | NOVEMBER 2005 | 837
© 2005 Nature Publishing Group http://www.nature.com/naturemethods
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